Mass fingerprinting of the venom and transcriptome of venom gland of scorpion Centruroides tecomanus

Abstract

Centruroides tecomanus is a Mexican scorpion endemic of the State of Colima, that causes human fatalities. This communication describes a proteome analysis obtained from milked venom and a transcriptome analysis from a cDNA library constructed from two pairs of venom glands of this scorpion. High perfomance liquid chromatography separation of soluble venom produced 80 fractions, from which at least 104 individual components were identified by mass spectrometry analysis, showing to contain molecular masses from 259 to 44,392 Da. Most of these components are within the expected molecular masses for Na(+)- and K(+)-channel specific toxic peptides, supporting the clinical findings of intoxication, when humans are stung by this scorpion. From the cDNA library 162 clones were randomly chosen, from which 130 sequences of good quality were identified and were clustered in 28 contigs containing, each, two or more expressed sequence tags (EST) and 49 singlets with only one EST. Deduced amino acid sequence analysis from 53% of the total ESTs showed that 81% (24 sequences) are similar to known toxic peptides that affect Na(+)-channel activity, and 19% (7 unique sequences) are similar to K(+)-channel especific toxins. Out of the 31 sequences, at least 8 peptides were confirmed by direct Edman degradation, using components isolated directly from the venom. The remaining 19%, 4%, 4%, 15% and 5% of the ESTs correspond respectively to proteins involved in cellular processes, antimicrobial peptides, venom components, proteins without defined function and sequences without similarity in databases. Among the cloned genes are those similar to metalloproteinases.

Figure 1. HPLC separation of soluble venom.

Venom (2.3 mg) from C. tecomanus was separated in a C18 reverse-phase column using a linear gradient from solution A (water in 0.12% TFA) to 60% solution B (acetonitrile in 0.10% TFA) over 60 min.

Figure 2. Molecular mass distribution of separated venom.

The histogram shows the distribution of the 104 different molecular weights (MW), in Da, present in the venom of C. tecomanus. Peptides and proteins having MWs from 3.0, 4.0, 6.0 and 7.0 K Da are the most abundant components. The molecular weigths were obtained by LCQ Fleet and LTQ-Orbitrap-Veloz mass spectrometer (see materials and methods).

Figure 3. Transcripts from venom glands of C. tecomanus.

Relative proportion (percentage) of each category obtained from the analysis of the transcripts from C. tecomanus venom gland library.

Figure 4. Multiple sequence alignment of putative Na⁺-channel toxin of C. tecomanus.

Alignment of the 24 complete sequences of putative Na⁺-channel toxin of cDNA library from C. tecomanus. Amino acids identical in all sequences are indicated with an asterisk and the cysteines are shown shaded. The Ct11 and Ct26 sequences are identical at amino acid level but different at nucleotide level. The sequences of mature peptides Ct23 and Ct20 are identical, but the signal peptide of their respective precursors, are different. The amino acids that theoretically give rise to modification in the C-terminal (amidation) are shown in italics. The theoretical molecular weights were calculated taking into account these changes using the protparam program (http://web.expasy.org/protparam program).

Figure 5. Sequence alignments of putative K⁺-channel toxins of C. tecomanus.

A: sequence alignment of putative alpha-toxins found in the cDNA library of C. tecomanus, where the cysteines are shown shaded. The amino acids involved in a possible modification of the C-terminal are shown underlined (and in italics); the percentage of identity is indicated (% I). B: Sequence alignment of the putative β-toxin Ct30 of C. tecomanus with the sequence of TdiKIK toxin from Tityus discrepans (gb|ABE98264.1 |), which is the one showing the greatest identity. Identical amino acids are indicated in shade and are shown in bold. The number of amino acids (a.a) and the theoretical mass of each sequence of C. tecomanus are indicated. NA = Not applicable.